West Nile virus (WNV) has become the leading cause of viral encephalitis in the United States. In this application I propose to investigate modulation of the innate immune response by WNV, an NIAID category B priority pathogen. The innate immune system is the first line of defense against invading pathogens. Recognition of pathogen-associated molecular patterns (PAMP) by specific receptors leads to type I interferon (IFN) production, the establishment of an anti-microbial state and directs the orientation of the adaptive immune response. TLR-3 is a receptor for double stranded RNA (dsRNA), a PAMP produced during many viral infections, and activates IFN production through activation of IRF-3. We have demonstrated that in WNV-infected cells, activation of IRF-3, is inhibited in response to dsRNA. I hypothesize that WNV nonstructural proteins specifically interfere with TLR-3 signal transduction by inhibiting the activation of signaling components downstream of TLR-3. I further hypothesize that modulation of IRF-3 activation by WNV positively affects WNV replication. The following Specific Aims will be addressed: 1) Identify the viral factors that interfere with dsRNA signaling. 2) Determine cellular targets of WNV interference with dsRNA-induced signaling. 3) Determine the roles of IRF-3 activation pathways in WNV infection. IMPACT: Our results suggest that WNV has evolved a mechanism(s) to interfere with one of the first immune responses to infection. The significance of this inhibition is not understood but is likely to have important consequences for WNV pathogenesis. By identifying the viral factors responsible for this inhibition and their cellular targets, we will gain important insight into mechanisms that regulate activation of IRF-3, as well as identify new functions for WNV proteins aside from their role in supporting viral replication. The experiments proposed in this application describe a focused approach to understanding WNV modulation of this particular aspect of the innate immune response and will provide the basis for long-term studies that will involve investigations into the ramifications of this modulation for WNV pathogenesis. Results obtained from this study are likely to yield insights into innate immune regulatory mechanisms of great significance for WNV infection, as well as for infections by other viruses. [unreadable] [unreadable]